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Detecting reactive oxygen species (ROS) that play a critical role as redox modulators and signalling molecules in biological systems currently requires invasive methods such as ROS -specific indicators for imaging and quantification. We developed a non-invasive, real-time, label-free imaging technique for assessing the level of ROS in live cells and thawed cryopreserved tissues that is compatible with in-vivo imaging. The technique is based on autofluorescence multispectral imaging (AFMI) carried out in an adapted fluorescence microscope with an expanded number of spectral channels spanning specific excitation (365 nm-495 nm) and emission (420 nm-700 nm) wavelength ranges. We established a strong quantitative correlation between the spectral information obtained from AFMI and the level of ROS obtained from CellROX staining. The results were obtained in several cell types (HeLa, PANC1 and mesenchymal stem cells) and in live kidney tissue. Additioanly,two spectral regimes were considered: with and without UV excitation (wavelengths > 400 nm); the latter being suitable for UV-sensitive systems such as the eye. Data were analyzed by linear regression combined with an optimization method of swarm intelligence. This allowed the calibration of AFMI signals to the level of ROS with excellent correlation (R = 0.84, p = 0.00) in the entire spectral range and very good correlation (R = 0.78, p = 0.00) in the limited, UV-free spectral range. We also developed a strong classifier which allowed us to distinguish moderate and high levels of ROS in these two regimes (AUC = 0.91 in the entire spectral range and AUC = 0.78 for UV-free imaging). These results indicate that ROS in cells and tissues can be imaged non-invasively, which opens the way to future clinical applications in conditions where reactive oxygen species are known to contribute to progressive disease such as in ophthalmology, diabetes, kidney disease, cancer and neurodegenerative diseases., Competing Interests: Declaration of competing interest Authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)